Bile Salts: The production of bile is an important function of human and animal liver hepatocytes and plays a crucial role in hepatobiliary and intestinal homeostasis and digestion. [de Buy Wenniger, Bile salts and cholestasis. Digest Liver Diseases 42(6), 409-18, 2010] Bile comprises a highly concentrated solution of bile salts—also known as conjugated bile acids—biliary lipids (phospholipids and cholesterol) and electrolytes. [Kis et al., Effect of membrane cholesterol on BSEP/Bsep activity: specificity studies for substrates and inhibitors, Drug Metabolism and Disposition 37, 1878-1886, 2009] Bile salts are synthesized in the liver via a series of metabolic steps starting from cholesterol. Bile salts and acids are secreted into bile and stored in the gallbladder for release. [Einarsson et al., Bile acid formation in primary human hepatocytes, World journal of Gastroenterology 6(4), 522-525, 2000; Jansen and Faber, 2.3.6 Metabolism of bile acids in Hepatology—From Basic Science to Clinical Practice, Third edition, 2007, 174-181]
Bile salts or conjugated bile acids include glycocholic acid, taurocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid, glycolithocholic acid and taurolithocholic acid (see FIG. 1).
After a meal, the gallbladder contracts, and stored bile is secreted into the intestinal tract where it plays a key role in the absorption of dietary lipids, fat-soluble vitamins, nutrients, and some drugs and drug candidates. In the intestine, approximately 90-95% of secreted bile salts are reabsorbed and returned to the liver and taken up there by hepatocytes—a process called enterohepatic circulation. [Jansen and Faber id. page 174] Enterohepatic circulation serves an important physiological function not only for the recycling of bile salts and absorption of dietary lipids, fat-soluble vitamins, nutrients and some drugs and drug candidates, but also for the regulation of whole-body lipid metabolism. [Chiang, Bile acids: regulation of synthesis. Journal of Lipid Research 50, 1955-1966, 2009]
Bile salts are indispensable for the formation of bile flow; secretion of cholesterol and phospholipids from the liver, formation of mixed micelles that keep fat-soluble organic compounds in solution in the gut, promotion of the dissolution and hydrolysis of triglycerides by pancreatic enzymes, and act as signaling molecules in the regulation of enzymes and transporters of drugs and intermediary metabolism. [Jansen and Faber, id. page 178]
Biosynthesis of bile salts involves a multi-step process beginning with the initial oxidation of cholesterol by cytochrome P450 oxidase enzymes (also known as mixed function oxidases) present in human hepatocytes, (FIG. 2) [id. page 174, Chiang id. page 1955] Two main routes exist for the conversion of cholesterol to the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA):] a classic or neutral pathway involving initial oxidation by the cytochrome P450 CYP7A1 (cholesterol 7α-hydroxylase) and alternative or acidic pathway involving side chain hydroxylation with cytochrome P450 CYP27A1 (sterol 27-hydroxylase). [Jansen and Faber id. page 174] CYP7A1 is regarded as the rate-limiting enzyme in bile acid synthesis and deficiencies in animal models have been associated with severe liver failure. [Jansen and Faber id.] The CYP27A1 product is not a substrate fur CYP7A1 but instead is oxidized by another cytochrome P450 enzyme named CYP7B1. From there, the neutral and acidic pathways overlap producing CDCA and CA. Other minor oxidative pathways may also contribute to bile acid synthesis. [Jansen and Faber id.] Most bile acids, including CA and CDCA, are conjugated to the amino acids glycine (G) and taurine (T) by two enzymes: bile acid:CoA synthase (BACS) and bile acid:amino acid transferase (BAT). [Chiang id. page 1957] These glycine and taurine bile acid conjugates act to decrease the toxicity and increase the aqueous solubility of unconjugated bile acids for secretion into bile. [Chiang id. page 1957] In the intestine, the glycine- and taurine-conjugated CA and CDCA can be deconjugated releasing CA and CDCA, which can be acted on by gut bacterial 7α-dehydroxylase to remove their 7α-hydroxy groups and thereby produce the secondary bile acids deoxycholic acid (DCA; 3α-12-dihydroxy CA) and lithocholic acid (LCA; 3α-monohydroxy). [Chiang id.] CA, CDCA, and DCA can be reabsorbed in the intestine and transported back to the liver to inhibit bile acid synthesis. Most of LCA is excreted in feces. The reabsorbed bile acids are further conjugated to amino acids producing the bile salts of CA, CDCA, and DCA.
Amino acid conjugated bile acids are termed conjugated bile acids or bile salts while non-amino acid conjugated bile acids are termed free. Bile acids and salts can be potentially toxic to cells and their concentrations under physiological conditions are tightly regulated. As mentioned above, bile acids are important and potent signaling molecules in the liver and intestine. Both free and conjugated bile acids bind to the ligand-binding domain of the nuclear transcription factor farnesoid X receptor (FXR; NR1H4), regulating FXR and associated gene transcription product FGF19 and ultimately regulating bile acid synthesis, excretion, and transport. [Chiang id. page 1956] Additionally, free and conjugated bile acids have been found to bind and activate pregnane X receptor (PXR; NR1I2) and vitamin D receptor (VDR; NR1I1). [Chiang id.]
The process of producing bile salts essentially results in the conversion of the hydrophobic cholesterol molecule into an amphipathic molecule that can serve physiologically as a detergent for absorption and transport of nutrients, fat-soluble vitamins, drugs, and other chemicals.
Bile salts have important acid-base properties, especially in the intestinal duodenum where pH values range from 3 to 5 units. [Costanzo, Physiology, 4th edition, Saunders Elsevier, Philadelphia, Pa., 2010] Unconjugated bile acids have pKa values ranging near 7 pH units. In the duodenum, unconjugated bile acids are almost exclusively in the unionized protonated form and therefore are relatively insoluble in water and readily reabsorbed by the intestinal epithelium cells. Bile salts or conjugated bile acids have much lower pKa values ranging from 1 to 4 units whereas the conjugated bile salts are ionized or deprotonated in the duodenum, are more water soluble, and are more able to emulsify lipids and other non-water-soluble agents.
Bile salts or conjugated bile acids in the duodenum having been ionized, are not readily reabsorbed and can build up in concentrations to allow for formation of micelles and solubilized lipids which play significant roles in processes such as elimination of cholesterol from the body, removal of catabolites produced by the liver, and emulsifying lipids, fat-soluble vitamins, and some drugs and drug candidates. [Jansen and Faber id. page 178]
Hepatocytes: Hepatic parenchymal cells, or hepatocytes, are polyhedral or spherical in nature and account for approximately 60% of the cells in the liver; they represent 80% or more of the total liver volume. [de la Iglesia, Morphofunctional aspects of hepatic structure, Handbook of Drug Metabolism, edited by T. F. Woolf, 1999, page 83] Hepatocytes are polar in nature and one skilled in the art would recognize what is termed an apical (canalicular) membrane or domain and a basolateral (blood or sinusoidal domain) membrane or domain. The hepatocyte basolateral membrane or domain is involved in the uptake of drugs and xenobiotics into the cell, while the apical membrane or domain provides a route for intracellular produced bile salts to be excreted or transported into bile flow and eventually to the common bile duct for secretion into the intestine.
Hepatocytes have specialized transport systems or transcellular transporters located at the basolateral membrane and the apical membrane. [Morgan et al., Interference with bile salt export pump function is a susceptibility factor for human liver injury in drug development, Toxicological Sciences 118(2), 485-500, 2010] These hepatobiliary transporters maintain liver homeostasis by regulating intracellular exposure to endobiotic and xenobiotic chemicals. Transport systems comprising of specific transporter proteins have been extensively investigated. Transporters at the basolateral membrane are involved in hepatocellular uptake of various substrates from the blood and sinusoids, elimination to the blood and sinusoids, or both depending on the transporter. Transporters on the apical membrane, however, are exclusively efflux transporters, mediating secretion into the bile flow of various substrates including bile acids and salts. [Morgan et al., id. page 485]
Bile Salts Export Pump: BSEP (all capitalized letters reflect human transporter gene product) is a membrane-associated transporter protein located on the hepatocyte apical or canalicular membrane and is a member of the superfamily of ATP-binding cassette (ABC) transporter proteins, which are responsible for the extracellular transport or secretion of conjugated and unconjugated bile acids and salts into the bile canaliculi. [Kis et al., BSEP inhibition: in vitro screens to assess cholestatic potential of drugs, Toxicology In Vitro 26(8), 1294-9, 2012] BSEP is also known as ATP-binding cassette, sub-family B member 11, ABCB11which is the protein product of the human ABCB11 gene (italics reflect the human gene). BSEP was first cloned in 1998from rat and identified as the “sister of P-glycoprotein (sPGP)”, based on its close amino acid sequence similarity to P-glycoprotein. [Kis et al., Effect of membrane cholesterol on BSEP/Bsep activity: species specificity studies for substrates and inhibitors, Drug Metabolism and Disposition 37, 1878-1886, 2009, page 1878] BSEP displays higher transport affinity binding for tauro- and glycochenodeoxycholic acid and lower for taurocholic acid glycocholic acid and tauroursodeoxycholic acid. [Jansen and Faber id. page 178] BSEP can transport to a limited extent unconjugated bile acids. [id.] BSEP, in addition to exporting bile salts, can also export some xenobiotics and drugs including pravastatin and vinblastine. [Morgan et al., id. page 485]
Rat and mouse orthologs of the human BSEP have similar amino acid sequences sharing 82% and 80%, respectively. [Yabutichi H, et al, Cloning of the dog bile salt export pump (BSEP; ABCB11) and functional comparison with the human and rat proteins, Biopharmaceutical Drug Disposition, 29(8), 441-8, 2008] BSEP is specialized for transporting monovalent bile salts taurine and glycine conjugates—through the canalicular membrane against a concentration gradient in an ATP-dependent manner. [Kis et al., id. page 1878] BSEP transport of bile salts is a saturable process with Km values for bile salts in the low micromolar range. [Kis id.] The sensitivity to impairment in BSEP transport function appears to display species specificity. [Kis id.] Mutations in human BSEP can lead to progressive intrahepatic cholestasis and liver failure (see below).
Additional ABC transporters expressed at the apical and basolateral membrane include multidrug-resistance related protein MRP2 (ABCC2), breast cancer resistance protein BCRP (ABCG2, also known as MXR) and multidrug-resistance protein MDR1 (ABCB1, also known as P-glycoprotein). [Chandra and Brouwer, Pharmaceutical Research 21(5) 719-735, 2004]
In humans, the levels of the various transporter proteins are subject to genetic polymorphism in the encoding genes as well as in these transcription factors. Adverse drug reactions may be caused by genetic or disease-induced variations of transporter expression or drug-drug interactions at the level of these transporters. [Faber et al., Drug transport proteins in the liver, Advanced Drug Delivery Reviews 55(1), 107-24, 2003]
Drug-Induced Liver Injury (DILI): Drug-induced liver injury encompasses a spectrum of clinical diseases ranging from mild biochemical abnormalities to acute liver failure. [Hussanin and Farrington, Idiosyncratic drug-induced liver injury; an overview, Expert Opinion in Drug Safety 6(6), 673-84, 2007] Most frequently, the underlying mechanism of DILI is poorly understood. In some cases of DILI, the liver injury is categorized as idiosyncratic—unknown etiology. [Wolf et al., Use of cassette dosing in sandwich-cultured rat and human hepatocytes to identify drugs that inhibit bile acid transport, Toxicology In Vitro 24(1), 297-309, 2010; Lee, Drug-induced hepatotoxicity, New England journal of Medicine 349(5), 474-85, 2003] The incidence of DILI induced hepatotoxicity in clinically marketed drugs is relatively rare, ranging from 1 in 5,000 to 1 in 10,000 or less. This is particularly true for DILI that results in severe liver injury leading to irreversible liver failure that can be fatal or require liver transplantation. DILI is a major cause of removal of approved drugs from the United States market resulting in removal of clinically significant therapeutics from patients in need of such therapy. [FDA Guidance for Industry: Drug-induced liver injury—premarketing clinical evaluation, July 2009; Ansede et al., An in vitro assay to assess transporter-based cholestatic hepatotoxicity using sandwich-cultured rat hepatocytes. Drug Metabolism and Disposition 38, 276-280, 2010] Additional consequences of DILI include class action lawsuits against the innovator company (with multi-million of dollar settlements), while adding additional time, expense, and uncertainty to the drug discovery and development process.
Because the modern drug development process requires extensive preclinical testing of drug candidates and subsequent clinical trials, drugs that do ultimately lead to DILI are rare. Drug candidates that display a toxic potential are usually removed from development and never reach the market. [FDA Guidance id.] Nevertheless, drugs that later result in DILI do get approved. Reasons for this may involve the relatively rare nature of the adverse event and that clinical trials are conducted in a closely controlled patient environment with a limited number of subjects for a limited time. Following marketing approval, the number of individuals administered a therapeutic agent will be much greater, periods of treatment may be much longer, and patients are less well monitored. Individuals display a wide variability in hepatic function and can differ greatly with respect to inherent hepatic metabolic function, environmental factors and co-medications. Risk factors for DILI include age, sex and genetic polymorphisms of drug-metabolizing enzymes such as cytochrome P450. In patients with human immunodeficiency virus, the presence of chronic viral hepatitis increases the risk of antiretroviral therapy hepatotoxicity. [Hussaini and Farrington, id. Abstract]
The relatively low incidence rate of DILI creates difficulties in detecting and diagnosing it; both for tests used and for numbers of patients needed. There is no finding that indicates DILI with certainty, including liver biopsy. Because DILI may simulate any known liver disease, the histopathologic picture frequently is reported to be “compatible with” the clinical and laboratory information available, but is not often diagnostic. Therefore, the diagnosis of DILI is one of exclusion, in which sufficient clinical information must be gathered to rule out other possible causes of the abnormal findings. This diagnosis by exclusion requires collecting considerable data at the time of the acute clinical situation, a process that frequently is not well or thoroughly done, so that available information is inadequate to establish the likelihood of drug causality with any reasonable degree of confidence. [FDA Guidance, page 3-7]
In most controlled clinical trials, monitoring is done to detect hepatic injury by serum enzyme (typically aminotransferases) activity increases. Because risks associated with the new drug are unknown, caution has dictated that stopping rules be used to limit liver damage during the trial. For safety reasons, the drug may be stopped before the full implications of its possible toxicity can be determined. Extrapolation of such data, despite early withdrawal of the drug in many cases, is used to predict the likelihood of future severe toxicity when the drug is used clinically.
For interpreting data from patients exposed to drugs in clinical trials, there is a hierarchy of findings that indicate progressively severe liver injury, beginning with serum amino-transferase activities as the most frequently abnormal and most sensitive test. [FDA Guidance id] In many clinical trials of new drugs, up to 15% of study patients (or even more) may demonstrate mild elevations of alanine aminotransferase (ALT) or aspartate aminotransferase (AST) activities. The threshold required to consider either more frequent monitoring of blood levels, or stopping the drug, is variously placed at twice the upper limit of the normal or reference range (2×ULN), 3×ULN, or 5×ULN. Monitoring is typically performed on a monthly basis but may be shortened to biweekly or weekly if elevations in serum enzyme levels are noted. According to the FDA guidance on drug-induced liver injury: